Abstract
The Fus3-MAP kinase module is a conserved phosphorylation signal system in eukaryotes that responds to environmental stress and transduction of external signals from the outer membrane to the nucleus. Aspergillus flavus can produce aflatoxins (AF), which seriously threaten human and animal health. In this study, we determined the functions of Fus3, confirmed Ste50-Ste11-Ste7-Fus3 protein interactions and phosphorylation, and explored the possible phosphorylation motifs and potential targets of Fus3. The regulatory mechanism of Fus3 on the biosynthesis of AF was partly revealed in this study. AF production was downregulated in Δfus3, but the transcriptional expression of most AF cluster genes was upregulated. It is notable that the levels of acetyl-CoA and malonyl-CoA, the substrates of AF, were significantly decreased in fus3 defective strains. Genes involved in acetyl-CoA and malonyl-CoA biosynthesis were significantly downregulated at transcriptional or phosphorylation levels. Specifically, AccA might be a direct target of Fus3, which led to acetyl-CoA carboxylase activities were decreased in null-deletion and site mutagenesis strains. The results concluded that Fus3 could regulate the expression of acetyl-CoA and malonyl-CoA biosynthetic genes directly or indirectly, and then affect the AF production that relies on the regulation of AF substrate rather than the modulation of AF cluster genes. IMPORTANCE Aspergillus flavus is an important saprophytic fungus that produces aflatoxins (AF), which threaten food and feed safety. MAP (mitogen-activated protein) kanases are essential for fungal adaptation to diverse environments. Fus3, as the terminal kinase of a MAPK cascade, interacts with other MAPK modules and phosphorylates downstream targets. We provide evidence that Fus3 could affect AF biosynthesis by regulating the production of acetyl-CoA and malonyl-CoA, but this does not depend on the regulation of AF biosynthetic genes. Our results partly reveal the regulatory mechanism of Fus3 on AF biosynthesis and provide a novel AF modulation pattern, which may contribute to the discovery of new strategies in controlling A. flavus and AF contamination.